The identities of physiologically important substrates for protein kinase C isoforms are poorly understood for most, if not all, cells and tissues. The phorbol ester series of tumor promoters have been widely used in numerous biologic systems because of their high potency, their ability to influence the behavior of diverse cell types, and most importantly, their high affinity binding to protein kinase(s) C. It is likely, but not proven, that the heterogeneity of cellular responses to phorbol esters is due to the diversity of PKC isoforms expressed in various cells, the diversity of protein substrates that are phosphorylated, or both. The hypothesis to be tested in this proposal is that a specific membrane transport protein, the Na/K/C1 cotransporter (also called NKCC1), is an essential component of the signal transduction pathway leading to mitogenesis triggered by potent PKC activators such as tumor-promoting phorbol esters. A specific hypothesis to be tested is that direct phosphorylation of the cotransport protein at critical serine/threonine residue(s) results in altered function of the protein. In mouse epidermal keratinocyte cell lines, we specifically propose to do the following: 1. Derive cell lines with highly inducible NKCC1 expression, in order to ask whether upregulation of this putative target for PKC alters the cells' response to tumor promoters. 2. Test whether specific signal transduction pathways are the downstream effectors of PKC-regulated NKCC1, and ask whether isoform-specific dominant negative PKCs can ablate cellular responses to tumor promoters. 3. Determine whether NKCC1 is a direct substrate of PKC, which serine/threonine residues are phosphorylated, and test whether these residues are important for NKCC1 function. 3. Determine whether NKCC1 is a direct substrate of PKC, which serine/threonine residues are phosphorylated, and test whether these residues are important for NKCC1 function. It is a complex but important challenge to work out the details of signal transduction pathways that are activated in target cells or tissues following treatment with phorbol esters. Such knowledge could be the foundation for innovative approaches to cancer chemoprevention and/or therapies designed to interrupt tumor progression.